Processes and agents for detecting Listerias

ABSTRACT

The invention relates to agents and processes for detecting bacteria of the genus  Listeria,  in particular  L. monocytogenes.  The agents according to the invention include primers whose sequence is selected from the iap gene of  L. monocytogenes.  In addition, the agents according to the invention include peptides whose sequence is selected from the p60 protein and which are suitable for producing specific antibodies for the immunological detection of  L. monocytogenes.

This application is a divisional of Ser. No. 08/456,670, filed Jun. 1,1995, now U.S. Pat. No. 5,932,415, which is a divisional of Ser. No.08/412,227, filed Mar. 27, 1995, now abandoned, which is a continuationof Ser. No. 08/075,248, filed Jun. 11, 1993, now abandoned.

BACKGROUND OF THE INVENTION

The invention relates to agents and processes for detecting bacteria ofthe genus Listeria, in particular L. monocytogenes.

The members of the genus Listeria are gram-positive rod-shaped bacteriawhich occur ubiquitously. Seven different species belong to this genus:L. monocytogenes, L. Ivanovii, L. seeligeri, L. welshimeri, L. innocua,L. murrayi and L. grayi. Of these, only L. monocytogenes is pathogenicfor humans, endangering in particular the newly born, pregnant women andolder persons, as well as patients subjected to immunosuppression. L.monocytogenes infections frequently have a fatal outcome.

Contaminated foodstuffs, in which the organism can multiply even at lowtemperatures around 4° C., are frequently the cause of listeriainfections. Thus, various listeria epidemics have been attributed to theconsumption of contaminated food, such as, for example, raw milk, cheeseor coleslaw. Consequently, rapid detection processes for detectinglisterias, in particular in foodstuffs or clinical samples, are urgentlyrequired. These processes must additionally be able to distinguishbetween L. monocytogenes and the species which are not pathogenic forhumans. Furthermore, it must be possible to detect all variants of L.monocytogenes, which is the species which is pathogenic for humans.Recent discussions resulted in the proposal to use L. innocua asindicator organism for a potential contamination with L. monocytogenes.Therefore, the detection of L. innocua would be very useful as well.

Detection of L. monocytogenes is effected in a known manner usingprocesses which are based on culturing the microorganisms. The processdescribed in Int. J. Food Microbiol. 4 (1987), 249-256 takes two weeks.A somewhat faster process is recommended by the International DairyFoundation (IDF); however, it takes at least 6-8 days. Both processesare unsuitable for rapid identification because of the time they take.In addition, both processes are labor-intensive, since nutrient mediamust be inoculated repeatedly in order to obtain single colonies, andsince the isolates must subsequently be characterized using biochemicaland serological methods of investigations.

While the immunological tests which are currently on the market onlytake a few hours, they do not permit the important differentiationbetween different species of listerias. In these processes, also, atwo-day pre-enrichment cultivation is required.

A method is described in Appl. Environ. Microbiol. 54 (1988), 2933-2937in which L. monocytogenes is specifically detected using syntheticoligodeoxyribonucleotide probes. However, the probes which are used arenot sufficiently specific, since they also react with the species L.seeligeri, which is not pathogenic for humans. Prior multiplication ofthe organisms is required for this process as well: samples offoodstuffs, or their dilutions, are spread on agar plates, and then theinoculated plates are incubated and subsequently investigated by thecolony hybridization procedure using a radioactively labelled DNA probe.Detection takes place by autoradiography. This method, too, islabor-intensive and time-consuming.

The DNA sequence of the iap (invasion-associated protein) gene of L.monocytogenes is described in Infect. Immuno. 58, 1943-1950 (1990). Thisgene encodes a protein which is also known under the designation p60 andwhich occurs in variants in all Listeria species. In L. monocytogenesthis protein is responsible for the ability to invade animal cells. Apolynucleotide (400 bases) having a component sequence from this gene issuitable as a DNA probe for distinguishing L. monocytogenes from otherorganisms.

The polymerase chain reaction (PCR) permits the in vitro amplificationof nucleic acids, and prior cultivation is generally not necessary whenusing this process. In order to start the reaction, short nucleic acidfragments (primers) are required, which primers encompass the section ofthe genome which is to be amplified. Usually, two primers are required,each of which hybridizes with one nucleic acid strand. One of theprimers therefore possesses the complementary sequence to the relevantsection of the gene. The choice of these primers determines thespecificity of the detection reaction. The use of this process fordetecting L. monocytogenes is described in Appl. EnvironmentalMicrobiology 57, 606-609 (1991), in Letters Appl. Microbio. 11, 158-162(1990) and in J. Appl. Bact. 70, 372-379 (1991). More extensiveinformation regarding the details of these processes is available inthese publications. The DNA primers bind to the gene for listeriolysin,the listeria hemolysin. The specificity of these primers is at leastuncertain, as is evident from comments in J. Appl. Bact. 70: L.seeligeri cannot be differentiated with certainty from L. monocytogenes.The unambiguous detection of L. monocytogenes has thus hitherto not beenpossible using the PCR technique.

Polyclonal antibodies against L. monocytogenes p60 also react with thep60 protein of other, non-pathogenic Listeria species. Such antibodiesare therefore unsuitable for specifically detecting L. monocytogenes byimmunological processes. It is possible in principle to purify apolyvalent antiserum of this nature by the specific absorption ofinterfering antibody fractions: for this purpose, p60 protein from allthe other Listeria species is covalently bound to carriers. The unwantedantibody fractions can be specifically absorbed; an antiserum thenremains which only reacts with p60 protein from L. monocytogenes. Thismethod for obtaining an L. monocytogenes-specific serum is elaborate:substantial quantities of the polyvalent antiserum are required asstarting material, as are, in addition, the p60 iap gene products of thedifferent Listeria species. The obtention of monoclonal antibodiesagainst p60 protein would not be associated with this large materialrequirement; nevertheless, the raising of antibodies against particularepitopes depends on chance: it is first of all necessary to prepare alarge number of antibody-producing cell clones, from which suitableclones must then be selected. It has thus far not been possible toobtain antibodies in a targeted manner against epitopes which arespecific for L. monocytogenes. The same holds true for epitopes whichare specific for L. innocua.

SUMMARY OF THE INVENTION

It is the object of the present invention to provide improved agents andmethods for differentiating bacteria of the genus Listeria, inparticular for detecting bacteria of the species L. monocytogenes. Inparticular, primer sequences which are suitable for the PCR techniqueare provided according to the invention, as are peptides for thetargeted production of specific antibodies which are suitable for theimmunological detection of the species L. monocytogenes and L. innocua.

Upon further study of the specification and appended claims, furtherobjects and advantages of this invention will become apparent to thoseskilled in the art.

The invention relates to primers, selected from the iap gene, for theamplification of nucleic acids, for example by means of the polymerasechain reaction, characterized in that the primers contain, as acomponent sequence, at least one sequence according to one of theformulae Ia to Ih and/or an affiliated complementary sequence, it beingpossible for up to 20 further nucleotide moieties to be bound in frontof and/or behind this component sequence. Such primers are suitable fordetecting and differentiating bacteria of the genus Listeria, includingin particular the species L. monocytogenes, by means of PCR.

-   -   AATATGAAAAAAGC (SEQ ID NO:1) Ia    -   GCTTCGGTCGCGTA (SEQ ID NO:2) Ib    -   ACAGCTGGGATTGC (SEQ ID NO:3) Ic    -   ACTGCTAACACAGCT (SEQ ID NO:4) Id    -   TAACAGCAATTCAAG (SEQ ID NO:5) Ie    -   CTGAGGTAGCGAGC (SEQ ID NO:6) If    -   AGCACTCCAGTTGTTA (SEQ ID NO:7) Ig    -   GCAGTTTCTAAACCT (SEQ ID NO:8) Ih

In this context, primers are particularly preferred which contain asequence according to one of the formulae IIa to IIh and/or anaffiliated complementary sequence.

-   -   ATGAATATGAAAAAAGCAAC (SEQ ID NO:9) IIa    -   TTGGCTTCGGTCGCGTATAA (SEQ ID NO:10) IIb    -   GCTACAGCTGGGATTGCGGT (SEQ ID NO:11) IIc    -   CAAACTGCTAACACAGCTACT (SEQ ID NO:12) IId    -   CAATAACAGCAATTCAAGTGC (SEQ ID NO:13) IIe    -   TAACTGAGGTAGCGAGCGAA (SEQ ID NO:14) IIf    -   ACTAGCACTCCAGTTGTTAAAC (SEQ ID NO:15) IIg    -   CCAGCAGTTTCTAAACCTGCT (SEQ ID NO:16) IIh

The invention additionally relates to peptides which contain, as acomponent sequence, at least one sequence according to one of theformulae IIIa to IIIi, it being possible for in each case up to sevenamino acids to be bound by peptide linkages in front of and/or behindthis component sequence.

-   -   ProValAlaProThrGln (SEQ ID NO:17) IIIa    -   ThrGlnAlaThrThrProAla (SEQ ID NO:18) IIIb    -   AlaIleLysGlnThrAlaAsnThrAla (SEQ ID NO:19) IIIc    -   GlnGlnThrAlaProLysAlaProThr (SEQ ID NO:20) IIId    -   ValAsnAsnGluValAlaAlaAlaGluLysThrGlu (SEQ ID NO:21) IIIe    -   ThrProValValLysGlnGluValLys (SEQ ID NO:22) IIIf    -   ValLysGlnProThrThrGlnGlnThrAlaPro (SEQ ID NO:23) IIIg    -   IleLysGlnProThrLysThrValAlaPro (SEQ ID NO:24) IIIh    -   GlnGlnThrThrThrLysAlaProThr (SEQ ID NO:25) IIIi

In this context, peptides are particularly preferred which have asequence according to one of the FIGS. 2 a-i (SEQ ID NO:26-34,respectively), and FIGS. 5 a-d (SEQ ID NO:35-38, respectively).

The invention also relates to the use of one of the said peptides,having a component sequence according to one of the formulae IIIa toIIIi, for preparing immunogenic conjugates. Peptides having a sequenceaccording to one of the FIGS. 2 a-i and of the FIGS. 5 a-d areparticularly preferred for this purpose.

The invention also relates to an antibody which binds an epitope whichis formed from the polypeptide according to FIG. 3 (SEQ ID NO:39) orcontains a peptide according to one of the formulae IIIa-IIIi,preferably according to one of the FIGS. 2 a-i and of FIGS. 5 a-d.

The invention further relates to an antibody which can be prepared byimmunizing an experimental animal with a polypeptide according to FIG. 3or with an immunogenic conjugate which contains a peptide having 7 to 24amino acids selected from the polypeptide according to FIG. 3.

The invention also relates to a process for preparing an antibodydirected against the p60 protein from listerias by immunizing anexperimental animal with an immunogen and isolating the antibodies,characterized in that a polypeptide according to FIG. 3 or animmunogenic conjugate which contains a polypeptide according to FIG. 3is used as the immunogen. In this context, immunogenic conjugates arepreferred which contain a peptide having 7 to 24 amino acids selectedfrom the polypeptide according to FIG. 3, or which contain a peptideaccording to one of the formulae IVa-IVi, in which

-   -   X³ and X⁴ are each independently of one another hydrogen, an        arbitrary amino acid or an arbitrary oligopeptide having up to 7        amino acids.    -   X³ProValAlaProThrGlnX⁴ (SEQ ID NO:17) IVa    -   X³ThrGlnAlaThrThrProAlaX⁴ (SEQ ID NO:18) IVb    -   X³AlaIleLysGlnThrAlaAsnThrAlaX⁴ (SEQ ID NO:19) IVc    -   X³GlnGlnThrAlaProLysAlaProThrX⁴ (SEQ ID NO:20) IVd    -   X³ValAsnAsnGluValAlaAlaAlaGluLysThrGluX⁴ (SEQ ID NO:21) IVe    -   X³ThrProValValLysGlnGluValLysX⁴ (SEQ ID NO:22) IVf    -   X³ValLysGlnProThrThrGlnGlnThrAlaProX⁴ (SEQ ID NO:23)IVg    -   X³IleLysGlnProThrLysThrValAlaProX⁴ (SEQ ID NO:24) IVh    -   X³GlnGlnThrThrThrLysAlaProThrX⁴ (SEQ ID NO:25) IVi

Especially preferred are peptides having a sequence according to one ofthe FIGS. 2 a-i or 5 a-d.

The invention further relates to the use of a primer, which contains acomponent sequence according to one of the formulae (Ia-Ih or preferablya sequence according to one of the formulae IIa-IIh or an affiliatedcomplementary sequence, for detecting bacteria of the genus Listeria.

The invention also relates to processes for detecting bacteria of thegenus Listeria by means of a primer which contains a component sequenceaccording to one of the formulae Ia-Ih or preferably a sequenceaccording to one of the formulae IIa-IIh or an affiliated complementarysequence.

The invention further relates to the use of an antibody which isdirected against an epitope from the polypeptide sequence according toFIG. 3, or which is directed against one of the epitopes having an aminoacid sequence according to one of the FIGS. 2 a-i or of the FIGS. 5 a-d,for detecting bacteria of the genus Listeria.

The invention also relates to processes for detecting bacteria of thegenus Listeria by means of an antibody which is directed against anepitope from the polypeptide sequence according to FIG. 3, or which isdirected against one of the epitopes having an amino acid sequenceaccording to one of the FIGS. 2 a-i, or to one of the FIGS. 5 a-d.

The invention finally relates to test kits for detecting bacteria of thegenus Listeria, in particular of the species L. monocytogenes, by meansof the amplification of nucleic acids, for example by means of thepolymerase chain reaction, which contain a primer having a componentsequence according to one of the formulae Ia-Ih or preferably having asequence according to one of the formulae IIa-IIh, or an affiliatedcomplementary sequence.

The invention furthermore relates to test kits for the immunologicaldetection of bacteria of the species Listeria monocytogenes, in which anantibody which is directed against an epitope from the polypeptidesequence according to FIG. 3, or which is directed against one of theepitopes having an amino acid sequence according to one of the FIGS. 2a-i, is contained, as well as to test kits for the immunologicaldetection of bacteria of the species Listeria innocua, in which anantibody, which is directed against an epitope having an amino acidsequence according to one of the FIGS. 5 a-d is contained.

BRIEF DESCRIPTION OF THE DRAWINGS

Various other objects, features and attendant advantages of the presentinvention will be more fully appreciated as the same becomes betterunderstood when considered in conjunction with the accompanying drawing,in which like reference characters designate the same or similar partsthroughout the several views, and wherein:

FIG. 1 shows the result of the electrophoretic separation ofamplification products; the experimental details are presented inExample 8.

FIGS. 2 a-i show the amino acid sequences (SEQ ID NO:26-34 respectively)of the particularly preferred immunogenic peptides selected from thesequence of the p60 protein from Listeria monocytogenes.

FIG. 3 shows the amino acid sequence (SEQ ID NO:39) of the polypeptideselected from the sequence of the p60 protein from Listeriamonocytogenes, whose epitopes are suitable for the immunologicaldetection of bacteria of the genus Listeria.

FIG. 4 shows, for comparative purposes, the amino acid sequence (SEQ IDNO:40) of the p60 protein from Listeria monocytogenes, which ispresented in two component FIGS. a and b.

FIGS. 5 a-d show the amino acid sequences (SEQ ID NO:35-38 respectively)of the particularly preferred immunogenic peptides selected from thesequence of protein p60 from Listeria innocua.

The invention is described below in more detail. In this context, thedetails of biochemical, immunological and molecular biologicalprocesses, which are known to the person skilled in the art and whosedetails are described in the literature, are presumed. In theseprocesses, use can also be made of variations which are known per se butwhich are not described in detail here.

The oligonucleotides according to the invention described by theformulae Ia-Ih and IIa-IIh are suitable as primers for nucleic acidamplification methods, and thus for the specific detection of bacteriaof the genus Listeria. Their sequences are presented in the customarymanner, i.e., written from the 5′ end to the 3′ end. Depending on therequirements of the amplification system being used on any particularoccasion, either deoxyribonucleotides or ribonucleotides having thesequences according to the invention are employed. In the latter case,the thymidine moieties are on each occasion replaced by uridinemoieties. It is further known to the person skilled in the art thatfrequently the exchange of one or of a few bases in a nucleic acidsequence does not alter its biological properties. For this reason, thenucleotide sequences according to the invention also comprise thosewhich are derived by base exchange from the sequences Ia-Ih and IIa-IIh,and which biologically show the same effect as the respective primerhaving the original sequence. Since normally one primer should in eachcase react with one of the DNA strands, one of the primers is employedin the complementary sequence. The complementary sequence is obtained ina known manner according to the rules for base pairing.

Based on the respective sequence, the oligonucleotides according to theinvention can be synthesized by processes known to the person skilled inthe art, for example by the phosphotriester or the phosphoamiditemethod. The phosphoamidite method is preferably employed, in particularusing mechanized synthesizers. The method is described in TetrahedronLett. (1981) 22:1859-1862. Further details of synthetic processes ofthis nature are described, for example, in Winnacker, E. L. (1985) Geneund Klone [Genes and Clones], page 44-61 (VCH-Verlagsgesellschaft mbH,Weinheim).

The primers according to the invention are suitable for DNAamplification, for example using the polymerase chain reaction (PCR).For this purpose, the DNA is first dissociated into the single strandsby heating. Two primers are used which in each case hybridize with thehomologous DNA segment on one of the DNA strands in each case. Thegenome segment which lies between these two primers is amplified. Theprimers attached to the DNA represent the starting points for theamplification. A polymerase, preferably Taq DNA polymerase, subsequentlycompletes, in the presence of the four nucleoside triphosphates, thesecond strand corresponding to the sequence of the original DNA.Subsequently, the double strands which have arisen are dissociated onceagain into the single strands by heating. This amplification cycle canbe repeated a number of times. After a sufficient number ofamplification cycles, the amplified nucleic acid can be detected bymeans of known methods. For this purpose, the DNA can be separated bymeans of electrophoresis, and subsequently stained with ethidiumbromide, and finally detected by fluorescence using UV excitation.Detection using DNA hybridization is also possible. The details ofsuitable amplification and detection methods are also described inreview articles, e.g., Innis et al. (eds.), PCR Protocols (AcademicPress, Inc., Harcourt Brace Jovanovich, Publishers). Other nucleic acidamplification processes in which the primers according to the inventioncan be used are also known from the literature. These include the ligasechain reaction, described by Bond, S. et al. (1990), pp. 425-434, RavenPress (New York, N.Y./USA)).

The selection of the primers according to the preferred formulae IIa-IIhdetermines the position of the start points on the iap gene, and thusthe specificity of the detection reaction: thus, combinations of primersselected from the sequence of the iap gene proved to be unspecific, andconsequently unsuitable for detecting listerias by means of DNAamplification (in this connection see, for example, column F in Table1). However, other selected combinations proved to be specific for thegenus Listeria, others for groups of Listeria species, and others againfor individual Listeria species. Altogether, therefore, the selectionand the composition of the primers is critical. The selection of one ofthe two primers is always particularly critical, while the second primercan be more easily varied without significantly altering the specificityof the detection reaction. Consequently, according to the teaching ofthe present invention, for this second primer, a sequence can perfectlywell be chosen which does not correspond to one of the formulae Ia-Ih orIIa-IIh.

According to the invention, at least one of the primers is selected fromthe formulae Ia-Ih or preferably from the formulae IIa-IIh. As alreadyexplained, the second primer has substantially less influence than thefirst primer on the specificity of the amplification reaction. However,combinations are preferred in which both primers are selected from theformulae Ia-Ih or IIa-IIh. Examples of preferred combinations of thisnature are (typical results are summarized in Table 1):

a) When using a combination of a primer according to formula IIc with aprimer with the complementary sequence according to formula IIb, onlythe DNA of listerias is amplified, and not the DNA of other types ofbacteria (see column D in Table 1).

b) When using a combination of a primer according to formula IId with aprimer with the complementary sequence according to formula IIb, onlythe DNA of L. monocytogenes is amplified, and not the DNA of otherlisterias or other bacteria (see column B in Table 1).

c) When using a combination of a primer according to formula IIf with aprimer with the complementary sequence according to formula IIb, onlythe DNA of particular Listeria species is amplified, namely that of L.seeligeri, L. welshimeri and L. ivanovii, exclusively. This consequentlypermits group-specific detection (see column E in Table 1).

d) Another example of group-specific detection consists in the use of acombination of a primer according to formula IIh with a primer with thecomplementary sequence according to formula IIb: only the DNA of L.grayi and L. murrayi is amplified (see column G in Table 1).

e) Since the amplification products of different Listeria speciesexhibit varying molecular weights, bacteria of the genus Listeria can bedifferentiated by a combination of several primers (according toformulae IId, IIf, IIg and IIh) with the complementary sequence offormula IIb using one single polymerase reaction. Details of thisfurther development of the polymerase technique are evident from Example8 (see column H in Table 1, as well as FIG. 1).

As already mentioned, it is also possible to detect the amplificationproducts by nucleic acid hybridization. To do this, suitable nucleicacid fragments (nucleic acid probes) are added to the reaction mixtureafter the amplification. The nucleic acid probes possess a base sequencewhich is completely or partially complementary to the amplified genesegment. In addition, these probes are labelled for a detectionreaction: they can contain radioactive isotopes, or carry fluorescentlabels, or else be labelled by enzymes. Suitable labelling agents,methods for their introduction into the nucleic acid probe, anddetection methods, are known to the person skilled in the art.

In particular, the amplification reaction can be designed specificallyfor the genus Listeria (as described in more detail above under a)) orfor a group of Listeria species (as described above under c) and d)). Byusing nucleic acid probes which are in each case specific for onespecies, the presence of these species of Listeria can then be discernedin the reaction mixture. If the probes contain different labellingagents, different species can also be detected side by side. Thisvariation of the process consequently permits, in a similar manner tothat described above under e), the detection of different Listeriaspecies side by side.

The use of a nucleic acid probe, or of a mixture of different probes,which react with amplification products of all the Listeria speciesmakes it possible to check the specificity of the amplification reactionor to prepare a unitary detection reagent for different Listeriaspecies.

The peptides according to the invention, according to formulae IVa-IViand according to FIGS. 2 a-i or to FIGS. 5 a-d, can be incorporated intoimmunogenic conjugates. Using these conjugates, antibodies can beproduced which make it possible specifically to detect bacteria of thegenus Listeria using immunological methods.

The positions of the peptides according to the invention in the overallsequence of the p60 protein from Listeria monocytogenes are given below:

a) The sequence according to formula IIIa begins with proline atposition 148 of the p60 sequence (FIG. 4 a); the peptides according toFIGS. 2 a, 2 e and 2 f are also located in this region.

b) The sequence according to formula IIIb begins with threonine atposition 178 of the p60 sequence (FIG. 4 a); the peptides according toFIGS. 2 b and 2 h are also located in this region.

c) The sequence according to formula IIIc begins with alanine atposition 243 of the p60 sequence (FIG. 4 a); the peptides according toFIGS. 2 c and 2 i are also located in this region.

d) The sequence according to formula IIId begins with glutamine atposition 292 of the p60 sequence (FIG. 4 b); the peptide according toFIG. 2 d is also located in this region.

e) The sequence according to formula IIIe begins with valine at position71 of the p60 sequence (FIG. 4 a); the peptide according to FIG. 2 g isalso located in this region.

The sequences of the peptides according to the invention which are shownin FIGS. 5 a-d are derived from the total sequence of protein p60 fromListeria innocua; the same holds true for the partial sequence shown informula IIIf-i and IVf-i.

It is known to the person skilled in the art that the exchange of one orof a few amino acids in a peptide frequently does not alter itsbiological properties. For this reason, the peptide sequence accordingto the invention also comprise those which are derived, by amino acidexchange, from the sequences according to FIGS. 2 a-i, according toFIGS. a-d, or according to FIG. 3, and which biologically show the sameeffect as the respective peptides having the original sequence. One ofskill in the art can routinely determine preferred exchanges inaccordance with substitutions generally recognized as being preferred,e.g., according to the groups outlined in Dayhoff, M. O., Atlas ofProtein Sequence and Structure, Vol. 5, p. 98 (1972), and updatesthereof.

The selection of the peptides according to the invention proves to becritical. For example, when a particular peptide,

-   -   ThrAsnThrAsnThrAsnThrAsnThrAsnThrAsn SEQ ID NO:41 which is        encoded by a gene segment around nucleotide 1390 which is        specific for L. monocytogenes, was selected for the production        of antibodies, none of the antisera, surprisingly, showed a        reaction with the p60 protein.

Based on the sequence of the amino acids, the peptides can besynthesized by processes which are known to the person skilled in theart, for example by the t_(boc) or by the f_(moc)(tert-butyloxycarbonyl, or 9-fluorenylmethyloxycarbonyl) processes.Details of these processes are described, for example, in J. Am. Chem.Soc. 85, 2149-2154 (1963) and in Synthetic Polypeptides as Antigens (vanRegenmortel et al. (eds.), Elsevier 1988 (volume 19 of the seriesLaboratory Techniques in Biochemistry and Molecular Biology). Thef_(moc) process is preferred, in particular mechanized processvariations thereof. Details of the process, as well as suitable aminoacid protective groups, are known to the person skilled in the art.

Peptides are generally not suitable for producing antibodies. However,if peptides are coupled to high-molecular weight carrier substances,immonogenic conjugates are formed. The peptides according to theinvention can be conjugated with known carrier substances. Among theseare polyethylene glycols, serum albumins, KLH (keyhole limpethemocyanin), ovalbumin, glucose dehydrogenase from Bacillus megateriumand PPD (purified protein derivative of tuberculin). Preferred carriersubstances are KLH and glucose dehydrogenase from B. megaterium.

Besides this, bridging compounds (linkers) are frequently employed aswell. These are low-molecular weight organic compounds having at leasttwo linkable functional groups. Suitable compounds are known to theperson skilled in the art; among these are, for example,1,2-diaminoethane, succinic acid, β-alanine, 1,6-diaminohexane,6-aminocapronic acid, adipic acid and cysteine. Cysteine is preferablyemployed as the linker, with this amino acid residue being incorporatedduring the synthesis of the peptide. Linkers which contain both an aminoand a carboxyl function (e.g., β-alanine, 6-aminocaproic acid orcysteine), can be linked either at the C-terminus or at the N-terminusof the peptide. m-Maleimidobenzoic acid N-hydroxysuccinimide ester (MBS)is preferably employed for preparing the bonds between the peptide andthe carrier substance.

The said immunogenic conjugates serve to produce antibodies inexperimental animals according to known processes. Usually, mammals areused for this purpose, for example sheep, goats, rabbits or mice.Rabbits are preferred for producing polyclonal antibodies. However, itis also possible to produce monoclonal antibodies using the immunogenicconjugates according to the invention (SEQ ID NO:41).

Details of the immunological processes are known to the person skilledin the art. In addition, instructions for carrying out these processesare readily available in the literature; the following may be mentionedby way of example:

-   -   Antibodies, E. Harlow and D. Lane, Cold Spring Harbor (1988)    -   Woodard, L. F. and Jasman, R. L. (1985) Vaccine 3, 137-144    -   Woodard, L. F. (1989) Laboratory Animal Sci. 39, 222-225    -   Handbook of Experimental Immunology, Weir, D. M. et al. eds.        (1986): Blackwell Scientific Publications, Oxford, GB.

Among these processes are, for example, the conjugation and immunizationprocesses, as well the preparation and purification of antibodies, andalso immunological detection processes. The immunological detectionprocesses in which antibodies according to the invention can be usedinclude preferably agglutination processes, immunometric detectionprocesses, the immunoblot processes and in particular the sandwich ELISAprocesses.

According to the invention, the concept antibodies embraces bothimmunoglobulins and antisera. It is furthermore known to the personskilled in the art that, instead of a single antibody which is directedagainst a single epitope, a mixture of different antibodies of differingspecificity may frequently be used. This results in advantages, inparticular with regard to the sensitivity of detection. This applies inparticular to monoclonal antibodies, but also to other antibodies, whichare in each case directed against one epitope. Correspondingly, it canbe advantageous to combine a plurality of antibodies which are directedagainst different peptide structures according to the formulae IIIa-IIIior according to one of the FIGS. 3, 2 a-i or 5 a-d, for the useaccording to the invention and/or the processes according to theinvention.

Details regarding the preparation of the primers and peptides accordingto the invention, as well as of their use, are evident from thefollowing examples. The person skilled in the art will elicit furthermethodological details from the cited literature. The examples areintended to illustrate the subject of the invention, and do notrepresent any limitation of the invention.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius and unless otherwise indicated, allparts and percentages are by weight.

The entire disclosure of all applications, patents and publications,cited above and below, and of corresponding German applications P 42 19111.4 and P 42 39 567.4, are hereby incorporated by reference.

EXAMPLES Example 1 Preparation of the Primer According to Formula IIa

The primer according to formula IIa is prepared by the phosphoamiditemethod using the 380A DNA synthesizer from Applied Biosystems. Theessential features of the method are described in Tetrahedron Lett.(1981) 22:1859‥1862. Further details can be found in the literaturesupplied by the instrument manufacturer.

The primers according to formula IIc, IId, IIf, IIg and IIh are preparedin a corresponding manner. The primers according to formula IIb and IIeare prepared in the respective complementary sequence

(IIb: TTGGCTTCGGTCGCGTAGAATTCATA: (SEQ ID NO:10)

IIe: GCACTTGAATTGCTGTTATTG) (SEQ ID NO:43).

Example 2 Performance of the PCR Reaction for the Species-specificDetection of L. monocytogenes

A sample of bacteria containing about 1 μg of DNA, is suspended in 50 μlof buffer (10 mM Tris-HCl pH 8.5; 1.5 mM MgCl₂ and 50 mM KCl) and heatedat 110° C. for 5 minutes. Subsequently, primers according to formula IIdand IIe (see Example 1; in each case 0.4 μg), and 2.5 U of Taqpolymerase (from Pharmacia), dissolved in reaction buffer (10 mMTris-HCl pH 8.5; 1.5 mM MgCl₂ and 50 mM KCl), and in each case 200 μmolof dGTP, dATP, dTTP and dCTP are added (total reaction volume 100 μl).The first denaturation step lasts for 3 minutes at 94° C.

Subsequently, the reaction mixture is maintained at a temperature of 55°C. for 30 seconds (annealing phase), and at a temperature of 72° C. forone minute (elongation phase). The subsequent denaturation steps (at 94°C.) last for 45 seconds. After 30 reaction cycles, a concludingelongation step (at 72° C.), of 5-minute duration, is carried out.

The PCR products are separated on a polyacrylamide gel (6%) in a runningbuffer of Tris-borate (in each case 50 mM) containing EDTA (2.5 mM).Subsequently, the separated PCR products are stained with ethidiumbromide (0.1 mg/ml in water), and visualized by irradiation with UVlight (260 nm).

PCR products are only observed (see column A in Table 1) if DNA or cellsfrom L. monocytogenes are present in the sample.

Example 3 Performance of the PCR Reaction for the Species-specificDetection of L. monocytogenes

The process described in Example 2 is repeated using primers accordingto formula IId and IIb (see Example 1) instead of the primers accordingto formula IId and IIe. In this case, too, PCR products are onlyobserved in DNA or cells from L. monocytogenes are present in the sample(see column B in Table 1).

TABLE 1 Specificity of the polymerase chain reaction using differentprimers corresponding to formula IIa-IIh Combination: A B C D E F G HPrimer 1: IId IId IIg IIc IIf IIa IIh M³⁾ Reaction investigated Primer2:¹⁾ Bacteria: IIe IIb IIb IIb IIb IIb IIb IIb L. monocytogenes serovar1/2a EDG + + − + − + − + serovar 1/2a Mack.²⁾ + + − + − + − + serovar1/2b + + − + − + − + serovar 1/2c + + − + − + − + serovar 3a + + − + − +− + serovar 3b + + − + − + − + serovar 3c + + − + − + − + serovar 4a + +− + − + − + serovar 4ab + + − + − + − + serovar 4b + + − + − + − +serovar 4c + + − + − + − + serovar 4d + + − + − + − + serovar 4e + + − +− + − + serovar 7 + + − + − + − + L. ivanovii − − − + + + − + L.seeligeri − − − + + + − + L. innocua serovar 6a − − + + − + − + serovar6b − − + + − + − + serovar 4ab − − + + − + − + L. welshimeri − − − + + +− + L. murrayi − − − + − + + + L. grayi − − − + − + + + Enterococcusfaecalis − − − − − + − − Bacillus cereus − − − − − + − − Micrococcusflavus − − − − − + − − Legend: + PCR product detected − no PCR productdetectable ¹⁾Complementary sequence ²⁾Mack.: Mackaness strain ³⁾M:Mixture of primers accoding to formula IId, IIf, IIg and IIh;Amplification products can be differentiated on the basis of themolecular weight.

Example 4 Performance of the PCR Reaction for the Genus-specificDetection of Bacteria of the Genus Listeria

A sample of bacteria containing about 1 μg of DNA, is suspended in 50 μlwater and heated at 110° C. for 5 minutes. Subsequently, primersaccording to formula IIc and IIb (see Example 1; in each case 0.4 μg),and 2.5 U of Taq polymerase (from Pharmacia), dissolved in reactionbuffer (10 mM Tris-HCl pH 8.5; 1.5 mM MgCl₂ and 50 mM KCl), and in eachcase 200 μmol of dGTP, dATP and dCTP are added (total reaction volume100 μl). The first denaturation step lasts for 3 minutes at 9° C.Subsequently, the reaction mixture is maintained at a temperature of 56°C. for 30 seconds (annealing phase), and at a temperature of 72° C. for2 minutes (elongation phase). The subsequent denaturation steps (at 94°C.) each last for 45 seconds. After 30 reaction cycles, a concludingelongation step (at 72° C.), of 5-minute duration, is carried out.

The PCR products are separated on an agarose gel (1%) in a runningbuffer of Tris-borate (in each case 50 mM) containing EDTA (2.5 mM).Subsequently, the separated PCR products are stained by staining withethidium bromide (0.1 mg/ml in water), and visualized by irradiationwith UV light (260 nm).

In this case, PCR products are observed if DNA or cells from bacteria ofthe genus Listeria are present in the sample (see column D in Table 1).

Example 5 Performance of the PCR Reaction for the Group-specificDetection of listerias

A sample of bacteria containing about 1 μg of DNA, is suspended in 50 μlwater and heated at 110° C. for 5 minutes. Subsequently, primersaccording to formula IIf and IIb (see Example 1; in each case 0.4 μg),and 2.5 U of Taq polymerase (from Pharmacia), dissolved in reactionbuffer (10 mM Tris-HCl pH 8.5; 1.5 mM MgCl₂ and 50 mM KCl), and in eachcase 200 μmol of dGTP, dATP, dTTP and dCTP are added (total reactionvolume 100 μl). The first denaturation step lasts for 3 minutes at 94°C. Subsequently, the reaction mixture is maintained at a temperature of58° C. for 45 seconds (annealing phase), and at a temperature of 72° C.for one minute (elongation phase). The subsequent denaturation steps (at94° C.) last for 45 seconds. After 30 reaction cycles, a concludingelongation step (at 72° C.), of 5-minute duration, is carried out.

The PCR products are separated on an agarose gel (1%) in a runningbuffer of Tris-borate (in each case 50 mM) containing EDTA (2.5 mM).Subsequently, the separated PCR products are stained by staining withethidium bromide (0.1 mg/ml in water), and visualized by irradiationwith UV light (260 nm).

In this case, PCR products are observed only if DNA or cells of bacteriafrom the group L. ivanovii, L. seeligeri and L. welshimeri are presentin the sample (see column E in Table 1).

Example 6 Performance of the PCR Reaction for the Species-specificDetection of L. Innocua

A sample of bacteria containing about 1 μg of DNA, is suspended in 50 μlof buffer (10 mM Tris-HCl pH 8.5; 1.5 mM MgCl₂ and 50 mM KCl) and heatedat 110° C. for 5 minutes. Subsequently, primers according to formula IIgand IIb (see Example 1; in each case 0.4 μg), and 2.5 U of Taqpolymerase (from Pharmacia), dissolved in reaction buffer (10 mMTris-HCl pH 8.5; 1.5 mM MgCl₂ and 50 mM KCl), and in each case 200 μmolof dGTP, dATP, dTTP and dCTP are added (total reaction volume 100 μl).The first denaturation step lasts for 3 minutes at 94° C. Subsequently,the reaction mixture is maintained at a temperature of 62° C. for 60seconds (annealing phase), and at a temperature of 72° C. for 45 seconds(elongation phase). The subsequent denaturation steps (at 94° C.) lastfor 45 seconds. After 30 reaction cycles, a concluding elongation step(at 72° C.), of 5-minute duration, is carried out.

The PCR products are separated on an agarose gel (1%) in a runningbuffer of Tris-borate (in each case 50 mM) containing EDTA (2.5 mM).Subsequently, the separated PCR products are stained by staining withethidium bromide (0.1 mg/ml in water), and visualized by irradiationwith UV light (260 nm).

PCR products are only observed if DNA or cells of L. innocua are presentin the sample (see column C in Table 1).

Example 7 Performance of the PCR Reaction for the Group-specificDetection of listerias

A sample of bacteria containing about 1 μg of DNA, is suspended in 50 μlof water and heated at 110° C. for 5 minutes. Subsequently, primersaccording to formula IIh and IIb (see Example 1; in each case 0.4 μg),and 2.5 U of Taq polymerase (from Pharmacia), dissolved in reactionbuffer (10 mM Tris-HCl pH 8.5; 1.5 mM MgCl₂ and 50 mM KCl), and in eachcase 200 μmol of dGTP, dATP, dTTP and dCTP, are added (total reactionvolume 100 μl). The first denaturation step lasts for 3 minutes at 94°C. Subsequently, the reaction mixture is maintained at a temperature of56° C. for 45 seconds (annealing phase), and at a temperature of 72° C.for 45 seconds (elongation phase). The subsequent denaturation steps (at94° C.) last for 45 seconds. After 30 reaction cycles, a concludingelongation step (at 72° C.), of 5-minute duration, is carried out.

The PCR products are separated on an agarose gel (1%) in a runningbuffer of Tris-borate (in each case 50 mM) containing EDTA (2.5 mM).Subsequently, the separated PCR products are stained by staining withethidium bromide (0.1 mg/ml in water) and visualized by irradiation withUV light (260 nm).

In this case, PCR products are only observed if DNA or cells of bacteriafrom the group L. grayi and L. murrayi are present in the sample (seecolumn G in Table 1).

Example 8 Performance of a Combined PCR Reaction for theSpecies-specific Detection of L. monocytogenes and of L. innocua and forthe Group-specific Detection of the Groups L. ivanovii/L. seeligeri/L.welshimeri and L. grayi/L. murrayi

A sample of bacteria containing about 1 μg of DNA, is suspended in 50 μlof buffer (10 mM Tris-HCl pH 8.5; 1.5 mM MgCl₂ and 50 mM KCl) and heatedat 110° C. for 5 minutes. Subsequently, a mixture of primers accordingto formula IId, IIf, IIg, IIh and IIb (see Example 1; in each case 0.4μg), as well as 2.5 U of Taq polymerase (from Pharmacia), dissolved inreaction buffer (10 mM Tris-HCl pH 8.5; 1.5 mM MgCl₂ and 50 mM KCl), andin each case 200 μmol of dGTP, dATP, dTTP and dCTP, are added (totalreaction volume 100 μl). The first denaturation step lasts for 3 minutesat 94° C. Subsequently, the reaction mixture is maintained at atemperature of 56° C. for 45 seconds (annealing phase), and at atemperature of 72° C. for one minute (elongation phase). The subsequentdenaturation steps (at 94° C.) last for 45 seconds. After 30 reactioncycles, a concluding elongation step (at 72° C.), of 5-minute duration,is carried out.

The PCR products are separated on a polyacrylamide gel (4%) in a runningbuffer of Tris-borate (in each case 50 mM) containing EDTA (2.5 mM). Inaddition, a nucleic acid mixture (for example the product resulting fromcleavage of Spp1 phage DNA by the restriction endonuclease EcoRI) isincluded as a molecular weight standard. Subsequently, the separated PCRproducts are stained by staining with ethidium bromide (0.1 mg/ml inwater), and visualized by irradiation with UV light (260 nm).

The presence of DNA or cells of bacteria from the species L.monocytogenes, from the species L. innocua, from the group L.ivanovii/L. seeligeri/L. welshimeri or from the group L. grayi/L.murrayi can be differentiated on the basis of the different molecularweights (see column H in Table 1, as well as FIG. 1).

Example 9 Synthesis of the Peptide

CysGlnGlnGlnThrAlaProLysAlaProThrGlu (SEQ ID NO:42)

The f_(moc) process (9-fluorenylmethyloxycarbonyl protective group) isused for the synthesis of the peptideCysGlnGlnGlnThrAlaProLysAlaProThrGlu (SEQ ID NO:42). This peptidecorresponds to a peptide of the formula IVd with an additionalN-terminal cysteine residue as linker. A peptide synthesizer fromApplied Biosystems is used for the synthesis; the process parameters arecontained in the instrument documentation.

A polymeric support with 4-(2′4′-dimethoxyphenylaminomethyl)phenoxygroups serves as the solid phase. The amino acids are employed asα-N-f_(moc) derivatives. Any reactive side groups contained in the aminoacids are masked by additional protective groups which may be eliminatedby hydrolysis with trifluoroacetic acid. The peptide bonds are producedby activating the carboxyl groups with diisopropylcarbodiimide. Theorder in which the amino acid derivatives are put in is determined bythe desired sequence.

In the first step of the synthesis cycle, the amino group on the solidphase, i.e., in the first cycle the amino groups of the4-(2′4′-dimethoxyphenylaminomethyl)phenoxy residue of the support,reacts with the carboxyl group of the incoming amino acid, which isemployed as the α-N-f_(moc) derivative, where appropriate with protectedside chains, and which is activated by diiopropylcarbodiimide, as doesthe α-amino group of the last amino acid to be attached in the followingcycles. Amino acid derivatives which have not reacted are washed outwith dimethylformamide. Subsequently, the f_(moc) group is eliminated bytreating with 20% (V/V) piperidine in dimethylformamide. The rest of theprotective groups remain unaltered during this reaction. Following theremoval of the α-N-protective group, the next reaction cycle can begin.Once the last amino acid corresponding to the envisaged sequence hasbeen added, the protective groups of the side chains and the bond withthe support resin are cleaved by acid hydrolysis with trifluoroaceticacid. The peptide is subsequently purified by high pressure liquidchromatography.

The remaining peptides with the sequences according to the invention arealso synthesized in accordance with the procedure described above.

Example 10 Conjugation of the Peptide

CysGlnGlnGlnThrAlaProLysAlaProThrGlu with glucose dehydrogenase (SEQ IDNO:42)

a) Derivatization of the glucose dehydrogenase: 30 mg of glucosedehydrogenase from Bacillus megaterium from Merck (Art. No. 13732) aredissolved in 4 ml of sodium phosphate buffer (50 mM; pH 8.0). 6.78 mg ofN-y-maleimidobutyryloxysuccinimide (from Calbiochem), dissolved in 50 μlof dimethyl sulfoxide, are added to 2.4 ml of this solution, and themixture is left to stand at room temperature for 30 minutes.Subsequently, the excess N-y-maleimidobutyryloxysuccinimide is separatedoff chromatographically by gel filtration on PD-10 (from Pharmacia).Following the chromatography, 3.5 ml of a solution of the activatedcarrier protein are obtained, having a concentration of 4.5 mg/ml.

b) Coupling with the peptide: 5.2 mg of the peptide, prepared accordingto Example 9 and dissolved in 1 ml of sodium phosphate buffer (50 mM; pH7.0), are added to 1.1 ml of the solution from the above step and themixture is left to stand at room temperature for 3 hours. Subsequently,the peptide which has not been bound is separated offchromatographically by gel filtration on PD-10 (from Pharmacia).Following the chromatography, 3.5 ml of a solution of the conjugate areobtained, having a concentration of 2.3 mg/ml.

Conjugates with other peptides corresponding to the present inventionare also prepared in accordance with the procedure described above.

Example 11 Production of Polyclonal Antibodies Against the Peptide

CysGlnGlnGlnThrAlaProLysAlaProThrGlu (SEQ ID NO: 42)

Two rabbits are in each case injected intramuscularly with an emulsionconsisting of 0.18 ml of conjugate from Example 10, 0.07 ml ofphosphate-buffered saline and 0.25 ml of an oil adjuvant (MISA, 50, fromSeppic, France). Booster injections of the same quantities are giventhree, five and seven weeks after the initial injection. One week afterthe last injection, the animals are killed and exsanguinated. After theblood has coagulated, the antiserum is obtained by centrifugation andsodium azide is added to give a final concentration of 0.02%. Theantiserum is stored frozen at −20° C.

Example 12 Production of Monoclonal Antibodies Against the Peptide

CysGlnGlnGlnThrAlaProLysAlaProThrGlu (SEQ ID NO: 42)

Two mice are in each case injected subcutaneously with an emulsionconsisting of 0.1 ml of conjugate from Example 10 and 0.1 ml oiladjuvant (MISA 50, from Seppic, France). Booster injections of the sameamounts are given two, four and six weeks after the initial injection.Three days after the last injection, the animals are killed and thespleen is isolated. The cells from the spleen are isolated by customaryprocesses and fused with a permanent murine cell line. Cell lines whichform antibodies against the peptide CysGlnGlnGlnThrAlaProLysAlaProThrGlu(SEQ ID NO: 42) are selected from the fusion products.

Example 13 Immunological Detection of L. monocytogenes

a) Pre-culture and centrifugation of the bacteria: 10 ml of CASO brothare inoculated with material from several colonies of L. monocytogenesand incubated at 30° C. overnight. Subsequently, 1 ml of the culture iswithdrawn in each case. The bacterial cells are removed bycentrifugation (13000 rpm).

b) Identification reaction: In each case 300 μl of the supernatants fromthe previous step are pipetted into the wells of a microtiter plate andincubated at 4° C. overnight. Subsequently, each well is washed threetimes with 100 μl of washing solution (9 g/l NaCl and 0.05% Tween 20 inwater) on each occasion. 100 μl of antiserum prepared according toExample 11 are now pipetted into each well and the plate is incubated atroom temperature for one hour. Each well is once again washed threetimes with 100 μl of washing solution on each occasion. 100 μl ofanti-rabbit antibody solution labelled with alkaline phosphatase (Art.No. A 8025, from Sigma) are then pipetted into each of the wells, andthe plate is incubated at room temperature for 30 minutes. Each of thewells is washed once again with 100 μl of washing solution and thebound, enzyme-labelled antibody is subsequently detected. To do this,200 μl of a buffer solution containing substrate is added to each well,and the plate is incubated at room temperature for 30 minutes. Thereaction is stopped by the addition of 100 μl of 2N NaOH solution (Art.No. 9136, from Merck) to each well, and the reaction product is renderedvisible. A yellow-orange coloration indicates the presence of L.monocytogenes.

Example 14 Specific Detection of L. monocytogenes Using Immunoblotting

Bacteria are pre-cultured as described in Example 13a), and the cellsare centrifuged off. The cells which have been centrifuged off are takenup and suspended in 1 ml of phosphate-buffered saline. 2 μl of thissuspension are pipetted on to a nitrocellulose membrane (Hybond C, 0.45μm, Art. No. RPN 283 C, from Amersham). Once the solution has dried in,the membrane is treated at room temperature for one hour with a solutionof bovine serum albumin (10 g/l) in phosphate-buffered saline. Adilution (1:200) of the antiserum obtained in Example 11 is preparedusing a solution of bovine serum albumin (10 g/l) and Tween 20 (0.5 g/l)in phosphate-buffered saline (antibody solution A), as is a furtherdilution (1:500) of peroxidase-labelled anti-rabbit antibody(anti-rabbit IgG, Art. No. 68-397; from ICN Immunobiologicals) using thesame diluent (HRP-antibody solution). The membrane is incubated at roomtemperature for one hour with antibody solution A, and subsequentlywashed three times with phosphate-buffered saline containing 0.05% Tween20. In order to detect the antibody binding, the membrane issubsequently incubated at room temperature for one hour withHRP-antibody solution and in each case washed three times with a) Tween20 (0.5 g/l) in phosphate-buffered saline, b) phosphate-buffered salineand c) Tris buffer (50 mM; pH 7.4; containing 200 mM NaCl). For thecolor reaction, a solution of 4-chloro-1-naphthol (3 mg/ml in methanol)is diluted with five volumes of Tris buffer (50 mM; pH 7.4; containing200 mM NaCl), and hydrogen peroxide is added (final concentration 0.1g/l). The membrane is incubated in this substrate solution. A blue-blackcoloration indicates L. monocytogenes.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

1. An isolated antibody which specifically binds to an epitope selectedfrom the group consisting of one of the following peptides: SEQ ID NO:17, 20, 26, 29, 30 and 31 of the p60 protein from pathogenic Listeriamonocytogenes and wherein said antibody does not react with p60 proteinfrom other non-pathogenic Listeria species.
 2. An isolated antibodywhich can be prepared by immunizing an experimental animal with apeptide selected from the group consisting of one of the followingpeptides: SEQ ID NO: 17, 20, 26, 29, 30 and 31, or with an immunogenicconjugate which comprises a peptide selected from the group consistingof one of the following peptides: SEQ ID NO: 17, 20, 26, 29, 30 and 31,wherein said antibody specifically binds to the p60 protein frompathogenic Listerina monocytogenes and does not react with p60 proteinfrom other non-pathogenic Listeria species.
 3. An isolated antibody ofclaim 1, which binds to an epitope of a peptide of SEQ ID NO:
 26. 4. Anisolated antibody of claim 1, which binds to an epitope of a peptide ofSEQ ID NO:
 29. 5. An isolated antibody of claim 1, which binds to anepitope of a peptide of SEQ ID NO:
 30. 6. An isolated antibody of claim1, which binds to an epitope of a peptide of SEQ ID NO:
 31. 7. Anisolated antibody of claim 2, wherein the peptide is SEQ ID NO:
 17. 8.An isolated antibody of claim 2, wherein the peptide is SEQ ID NO: 26.9. An isolated antibody of claim 2, wherein the peptide is SEQ ID NO:29.
 10. An isolated antibody of claim 2, wherein the peptide is SEQ IDNO:
 30. 11. An isolated antibody of claim 2, wherein the peptide is SEQID NO:
 31. 12. The antibody of claim 1 which is a polyclonal antibody.13. The antibody of claim 1 which is a monoclonal antibody.
 14. Theantibody of claim 2 which is a polyclonal antibody.
 15. The antibody ofclaim 2 which is a monoclonal antibody.
 16. A composition comprising atleast two monoclonal antibodies each of which binds to a peptideaccording to claim
 1. 17. An isolated antibody of claim 2, wherein saidanimal is a sheep, goat, rabbit or mouse.